Method of collecting duodenal specimen to detect upper digestive system disease without using pancreatic or bile stimulant

ABSTRACT

A method of collecting a specimen of the present invention is used in detecting upper digestive system disease. The method of collecting a specimen includes: a step of positioning sampling equipment in the duodenum of the subject into which duodenal juice is secreted, the sampling equipment being used to collect and store the duodenal juice; a step of collecting duodenal juice naturally secreted in the duodenum using the sampling equipment; and a step of stopping collection of the duodenal juice when collection quantity of the duodenal juice reaches a predetermined quantity which is 3.0 ml or less.

CROSS REFERENCE TO RELATED APPLICATION

This application is based upon and claims the benefit of priority fromthe prior provisional U.S. patent application Ser. No. 61/471,858, filedApr. 5, 2011, the entire content of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of collecting a specimen and amethod of diagnosing a subject to detect upper gastrointestinal disease.In particular, the present invention relates to a method of collecting aspecimen and a method of diagnosing a subject suitable for detection ofearly stages of cancers, such as pancreatic cancer, biliary tractcancer, liver cancer, and stomach cancer.

2. Description of the Related Art

Rapid advancements are being made in methods of examination for uppergastrointestinal diseases (for example, pancreatic cancer, biliary tractcancer, gallstones, cholecystitis, pancreatitis, liver cancer,cirrhosis, stomach cancer, and scirrhous stomach cancer). The prognosisis extremely poor particularly for pancreatic cancer and biliary tractcancer among the upper gastrointestinal diseases. A method ofexamination is required to enable detection of early stages of thesecancers.

Conventionally, research has been reported regarding a method ofexamination for early cancers in which pancreatic juice and bile areused as specimens. In the method of examination, cytological diagnosisand protein analysis are performed on these specimens. However, in thecase of collecting the pancreatic juice or the bile, it is required toinsert a catheter into the pancreatic duct or the bile duct. Doctors arerequired to acquire significant skill in catheter insertion. Inaddition, catheter insertion may cause pancreatitis as an accidentalsymptom. Therefore, not all doctors can easily perform the method ofexamination using pancreatic juice and bile.

A method of pancreatic juice collection is reported in which a catheteris not inserted into the pancreatic duct or the bile duct. Rather,duodenal juice is collected from the duodenum. The duodenal juicecontains pancreatic juice and bile discharged from the pancreas and thegall bladder into the duodenum. The duodenal juice is then examined.

For example, “Clinical Gastroenterology and Hepatology 2006; 4(6) pp.782-789” reports a method of detecting pancreatic cancer in which theinterleukin-8 (IL-8) protein concentration present in duodenal juicecontaining pancreatic juice is measured using the enzyme-linkedimmunosorbent assay (ELISA) method. In this method, an uppergastrointestinal endoscope is inserted into the patient after fasting. Astimulant of pancreatic juice secretion (secretin) is then administered.Subsequently, pancreatic juice secreted from the duodenal papilla iscollected from the duodenum. The pancreatic juice is collected for tenminutes using a catheter that has been threaded through the endoscope.Then, the duodenal juice is frozen for preservation. In addition, thereport indicates that a protease inhibitor is added to the collectedduodenal juice. It is reported that, as a result, pancreatic cancer canbe detected with high sensitivity.

U.S. Pat. No. 5,651,769 reports a method of diagnosing chronicpancreatitis in which duodenal juice is collected from the duodenumusing an endoscope. In this method, to prevent gastric juice from mixingwith the duodenal juice, a double-lumen catheter is used to collect theduodenal juice. The double-lumen catheter is capable of collecting thegastric juice and the duodenal juice separately. In a manner similar toa secretin test conducted in the conventional method, the patient isadministered secretin. Subsequently, the duodenal juice is collected for15 minutes at a time over a one-hour period.

However, in these methods, the patient is administered a stimulant ofpancreatic juice secretion (such as secretin). Therefore, the methodsare invasive to patients. In addition, the cost of examination is highdue to the cost of the required drugs.

In addition, the collection time for duodenal juice is very long (10minutes or 15 minutes at a time over a one-hour period). Because a largeamount of time is required for examination, the burden placed onpatients and doctors is also great.

Furthermore, no reports have been made regarding the timing at which theprotease inhibitor is added to the duodenal juice. Therefore, if theprotease inhibitor is added to the duodenal juice after the duodenaljuice has been collected for 10 minutes or 15 minutes at a time over aone-hour period, the proteins and cells to be measured are broken downby the protease activated in the duodenal juice while the duodenal juiceis being collected and accumulated. Therefore, detection accuracy ofproteins and cells may decrease.

SUMMARY OF THE INVENTION

The present invention has been achieved in light of the issues of theconventional methods of examination described above. In particular, anobject of the present invention is to simplify the method of examinationand improve examination accuracy.

According to an aspect of the present invention, a method of collectinga specimen to detect upper digestive system disease is provided. Themethod of collecting a specimen includes: a step of positioning samplingequipment in the duodenum of the subject into which duodenal juice issecreted, the sampling equipment being used to collect and store theduodenal juice; a step of collecting duodenal juice naturally secretedin the duodenum using the sampling equipment; and a step of stoppingcollection of the duodenal juice when a collection quantity of theduodenal juice reaches a predetermined quantity which is 3.0 ml or less.

In addition, according to another aspect of the present invention, amethod of diagnosing a subject to detect upper digestive system diseaseis provided. The method of diagnosing a subject includes: a step ofpositioning sampling equipment in the duodenum of the subject into whichduodenal juice is secreted, the sampling equipment being used to collectand store the duodenal juice; a step of collecting duodenal juicenaturally secreted in the duodenum using the sampling equipment; a stepof stopping collection of the duodenal juice when collection quantity ofthe duodenal juice reaches a predetermined quantity which is 3.0 ml orless; and a step of detecting concentration of a protein included in thecollected duodenal juice using an immunochromatography method.

According to an aspect of the present invention, a method of diagnosinga subject to detect upper digestive system disease is provided. Themethod of diagnosing a subject includes: a step of positioning samplingequipment in the duodenum of the subject into which duodenal juice issecreted, the sampling equipment being used to collect and store theduodenal juice; a step of collecting duodenal juice naturally secretedin the duodenum using the sampling equipment; a step of stoppingcollection of the duodenal juice when collection quantity of theduodenal juice reaches a predetermined quantity which is 3.0 ml or less;and a step of conducting examination for cytological diagnosis of thecollected duodenal juice.

In addition, according to another aspect of the present invention, amethod of collecting a specimen to detect upper digestive system diseaseis provided. The method of collecting a specimen includes: a step ofpositioning sampling equipment in the duodenum of the subject into whichduodenal juice is secreted, the sampling equipment being used to collectand store the duodenal juice; a step of collecting duodenal juicenaturally secreted in the duodenum using the sampling equipment; and astep of stopping collection of the duodenal juice within five minutesfrom the start of collection.

According to an aspect of the present invention, a method of diagnosinga subject to detect upper digestive system disease is provided. Themethod of diagnosing a subject includes: a step of positioning samplingequipment in the duodenum of the subject into which duodenal juice issecreted, the sampling equipment being used to collect and store theduodenal juice; a step of collecting duodenal juice naturally secretedin the duodenum using the sampling equipment; a step of stoppingcollection of the duodenal juice within five minutes from the start ofcollection; and a step of detecting concentration of a protein includedin the collected duodenal juice using an immunochromatography method.

In addition, according to another aspect of the present invention, amethod of diagnosing a subject to detect upper digestive system diseaseis provided. The method of diagnosing a subject includes: a step ofpositioning sampling equipment in the duodenum of the subject into whichduodenal juice is secreted, the sampling equipment being used to collectand store the duodenal juice; a step of collecting duodenal juicenaturally secreted in the duodenum using the sampling equipment; a stepof stopping collection of the duodenal juice within five minutes fromthe start of collection; and a step of conducting examination forcytological diagnosis of the collected duodenal juice.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a drawing showing the constitution of the sampling equipment;

FIG. 2 is an explanatory flowchart showing procedures of the method ofcollecting a specimen to detect upper digestive system disease accordingto an embodiment of the present invention;

FIG. 3 is an explanatory drawing showing the position from which theduodenal juice is collected;

FIG. 4 is a drawing showing the constitution of a collection system fora specimen in which the method of collecting a specimen according to anembodiment of the present invention is implemented;

FIG. 5A is a top view showing a fundamental structure of animmunochromatography device;

FIG. 5B is a perspective view showing a structure of detecting unitprovided inside the casing of an immunochromatography device;

FIG. 6 is a view showing average collection quantity and dispersion fordifferent collection times of duodenal juice collected in the 38patients of pancreatic disease;

FIG. 7 is a view showing CEA concentration every fractionation ofcollection about duodenal juice collected in the 38 patients ofpancreatic disease;

FIG. 8A and FIG. 8B are views showing S100P concentration in theduodenal juice accompanying the elapse of time, FIG. 8A is a view withno protease inhibitor added, and FIG. 8B is a view with proteaseinhibitor added;

FIG. 9A and FIG. 9B are views showing IL-8 protein concentration in theduodenal juice every fractionation of collection, FIG. 9A is a view inthe case of adding no protease inhibitor, and FIG. 9B is a view in thecase of adding a protease inhibitor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method of collecting a specimen and a method of diagnosing a subjectto detect upper digestive system disease of the present invention willhereinafter be described using examples, with reference to the drawings.

The upper digestive system disease that can be detected by the presentinvention can be any disease occurring in an organ in which fluidsecreted from the organ mixes with duodenal juice. Specifically, theupper digestive system disease includes diseases occurring in thepancreas or the biliary tract, diseases occurring in the liver thatproduces bile, and diseases occurring in the stomach. Diseases occurringin the pancreas or the biliary tract include, for example, pancreaticcancer, intraductal papillary mucinous neoplasm (IPMN), mucinous cysticneoplasm (MCN), chronic pancreatitis, acute pancreatitis, bile ductcancer, gallbladder cancer, cholangitis, cholecystitis, and gallstones.Diseases occurring in the liver that produces bile include, for example,liver cancer, cirrhosis, and hepatitis. Diseases occurring in thestomach include, for example, stomach cancer, scirrhous stomach cancer,submucosal tumors, and gastric ulcer. In the examples describedhereafter, pancreatic cancer and IPMN are detected.

Before procedures of the method of collecting a specimen for detectingupper digestive system disease are described, sampling equipment and anobservation device will be described.

(Sampling Equipment)

The sampling equipment is inserted into the body of a subject that is apatient. The sampling equipment collects duodenal juice and stores thecollected duodenal juice.

As shown in FIG. 1, the sampling equipment 1 is composed of a catheter 2for collection and a syringe 3. The syringe 3 is a collection containercapable of storing therein the duodenal juice. The syringe 3 isconnected to the catheter 2. Here, a tip end section of the catheter 2that is inserted into the subject first is referred to as a “tip 2A” ora “tip side 2A”. A section on the opposite side that is operated by anoperator is referred to as a “base 2B” or a “base side 2B”. The longerdirection of the linear shape of the catheter 2 is referred to as an“axial direction (in some instances, the longer direction)”. Thedirection of a plane perpendicular to the axial direction is referred toas a “radial direction”.

For example, an endoscopic retrograde cholangiopancreatography (ERCP)cannula PR-130Q (manufactured by Olympus Medical Systems Corporation)can be used as the catheter 2 for collection of the duodenal juice. Forthis collection, the cannula PR-130Q is provided with two holes 4 in theradial direction on the tip side 2A.

The syringe 3 houses a gasket 6 and a plunger 7 within a cylindricalouter casing 5. The gasket 6 is slidable in the longer direction of theouter casing 5. The plunger 7 is connected to the gasket 6. A flange 8is connected to the plunger 7 in the end section on the side opposite tothe side on which the gasket 6 is connected. The flange 8 is capable ofoperating the gasket 6 so as to move. A nozzle 9 is attached to theouter casing 5. The nozzle 9 is inserted into the end section on thebase side 2B of the catheter 2. A protease inhibitor is added in advanceinto a sealed space formed by the outer casing 5 and the gasket 6. Thecollected duodenal juice b is led through the catheter 2 into thesyringe 3.

The sampling equipment is not limited to the catheter 2 described in theabove example. The sampling equipment is merely required to be capableof being threaded through a forceps insertion opening of an endoscopeand collecting duodenal juice. Aspiration, absorption, collection, andthe like can be used as the collection method. However, the collectionmethod is not limited thereto. The collection container storing thereinthe duodenal juice b when the catheter is used is not limited to thesyringe 3. A collection trap set in front of the syringe 3 or acollection bottle connected to a vacuum pump may be used. As a result ofnegative pressure being generated within the catheter by the collectioncontainer, the duodenal juice b can be aspirated and stored within thecollection container.

(Observation Device)

The observation device is composed of an endoscope and a monitor. Theendoscope is inserted into the stomach or the duodenum and inputs animage of the interior of the organ. The monitor outputs the image fromthe endoscope.

The catheter 2 for collection is inserted into the forceps insertionopening of the endoscope and projected outward from the tip section ofthe endoscope. The tip of the endoscope is positioned using the imagedisplayed in the monitor. As a result, the position of the tip 2A of thecatheter 2 can be accurately decided.

The endoscope is merely required to be a commonly used endoscope. Forexample, a side-view endoscope often used in the biliopancreatic regionor an upper gastrointestinal endoscope used to examine the stomach andthe duodenum can be used. The insertion path of the endoscope may beoral or nasal.

The procedures of the method of collecting a specimen to detect upperdigestive system disease according to the present embodiment willhereinafter be described in time series with reference to FIG. 2.

As shown in FIG. 2, the following steps are performed.

(1) A stabilizer a for duodenal juice b is added in advance into thesyringe 3 serving as the sampling equipment 1 (Step 1).

The duodenal juice b contains a large quantity of pancreatic enzymeactivated by enterokinase in the duodenum. The pancreatic enzyme rapidlydegrades proteins and cells. Therefore, a protease inhibitor (such asethylenediaminetetraacetic acid [EDTA], aprotinin, phenylmethylsulfonylfluoride [PMSF], or 4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride [AEBSF]) or a pH-control chemical reagent that suppressespancreatic enzyme activity is preferably provided in advance, as thestabilizer a for duodenal juice b, in the syringe 3 connected to thebase side 2B of the catheter 2.

When collection of the duodenal juice b through the catheter 2 isstarted, the duodenal juice b is led into the syringe 3. Here, thestabilizer a for duodenal juice b is added into in the syringe 3 inadvance. Therefore, the duodenal juice b mixes with the stabilizer a forduodenal juice b. Pancreatic enzyme activity can be instantlysuppressed.

The stabilizer a for duodenal juice b may be in powder or liquid form.The stabilizer a for duodenal juice b preferably mixes quickly with thecollected duodenal juice b. Depending on the type of protein, describedhereafter, addition of the stabilizer a for duodenal juice b may not berequired. However, for most proteins, detection accuracy is improved bythe stabilizer a for duodenal juice b being added.

The protease inhibitor may be dispersed within the body. In thisinstance, a protease inhibitor that can be internally administered, suchas Futhan or aprotinin that can injected intravenously, can be used.

(2) Residual liquid present inside the stomach of the subject isaspirated (Step 2).

The throat of the patient after fasting overnight is anesthetized. Theendoscope is then inserted orally. At this time, use of a sedative agentor an antispastic agent is ordinarily preferred.

The endoscope is advanced from the esophagus to the stomach of thepatient. The gastric juice that has already oozed out into the stomachis then aspirated.

Here, washing of the stomach walls by sprinkling water from theendoscope is not actively performed. Therefore, changes in theconcentration to be measured (protein concentration) can be prevented.The changes in the concentration occur as a result of the gastric juiceand the fluid for washing the stomach flowing into the duodenum, andmixing with the duodenal juice b that is already accumulated.

(3) The sampling equipment 1 is placed in the duodenum of the subjectfrom which the duodenal juice b is secreted (Step 3).

The endoscope is advanced to the duodenum. When confirmation is obtainedvisually (via the camera connected to the endoscope) that the endoscopehas reached the duodenum, the catheter 2 for aspiration is inserted intothe forceps insertion opening of the endoscope and projected outwardfrom the tip section of the endoscope. Preparation for collecting theduodenal juice b is made.

(4) Naturally secreted duodenal juice b is collected using the samplingequipment 1 (Step 4).

Here, when collecting the duodenal juice b, a stimulant of pancreaticjuice secretion (such as secretin) that causes active secretion ofpancreatic juice and bile is not used. Rather, the duodenal juicenaturally accumulated in the duodenum is aspirated. The duodenal juice bcontains pancreatic juice, bile, and duodenal secretion and isconsidered to reflect information from each type of body fluid.

The position from which the duodenal juice b is collected will bedescribed. As shown in FIG. 3, pancreatic juice c is produced andsecreted by the pancreas 10. The pancreatic juice c accumulates in thepancreatic duct 11 running through the center of the pancreas 10. Bile dis secreted from the liver 12. The bile d is stored by the gallbladder13 positioned on the lower surface of the liver 12 and accumulates inthe common bile duct 15 connecting the gallbladder 13 and the duodenum14. The pancreatic duct 11 and the common bile duct 15 merge before theduodenum 14. The pancreatic juice c and the bile d are secreted into theduodenum 14 through the papilla of Vater 16.

Therefore, to collect the duodenal juice b, the tip 2A of the catheter 2is preferably placed near the papilla of Vater 16 that secretes thepancreatic juice c and the bile d. The duodenal juice b is preferablycollected from near the papilla of Vater 16. However, the position ofthe papilla of Vater 16 differs with the individual. The papilla ofVater 16 may be hidden behind a fold of mucosa or may not be visible inpictures due to anatomical position. In addition, depending on thecollection site, the mixture ratio of pancreatic juice c and bile d andmarker concentration may significantly differ from those of other sites.Therefore, the duodenal juice b from a plurality of sites is preferablycollected from the overall second position 14X and third position 14Y ofthe duodenum 14 shown in FIG. 3. The bile d is yellow. Therefore, alocation where yellow fluid is visible may be confirmed and collectionmay be performed. The duodenal juice b from the plurality of sites iscontinuously collected in the same collection container and mixed. Then,the collected duodenal juice b can be examined. As a result, a balancedexamination of duodenal juice b present within the body can beconducted. Examination accuracy and repeatability can be ensured.

(5) It is judged whether or not the collection quantity of duodenaljuice b meets a condition of 0.001 ml or more and 3.0 ml or less (StepS5).

Each subject has the timings at which the pancreatic juice c and thebile d are secreted into the duodenum 14. Conventionally, for collectingthe duodenal juice b, the timings are considered to differ depending oneach subject. Therefore, the duodenal juice b is collected over a longperiod of time after administration of the stimulant of pancreatic juicec secretion (such as secretin). However, the inventors of the presentinvention have found that proteins, described hereafter, can be detectedin high concentrations by collecting a small quantity of duodenal juiceb naturally secreted in the duodenum. The inventors have discovered thata small quantity of duodenal juice b can be sufficient as the specimen.

Here, the collection quantity of duodenal juice b is preferably aquantity that can be collected without damaging the mucosa of theduodenum 14. The collection quantity is a lower limit quantity or more,the lower limit quantity being that required for detecting theconcentration of a certain protein contained in the duodenal juice b orfor cytological diagnosis. In addition, the collection quantity is thequantity of duodenal juice b composed of pancreatic juice c, bile d, andduodenal juice b naturally secreted in the duodenum, or less.Specifically, the collection quantity of duodenal juice b is within arange of 0.001 ml or more and 3.0 ml or less, preferably 0.01 ml or moreand 1.0 ml or less, and more preferably 0.01 ml or more and 0.5 ml orless.

The inventors of the present invention have conducted a study onnumerous patients. The inventors have found that the quantity ofduodenal juice b naturally oozing into the duodenum 14 is about 2.0 ml.About 2.0 ml can be ordinarily collected when the duodenal juice b isaspirated for two to three minutes. The collection quantity is confirmedto not significantly increase when collection is continued beyond thisduration. In particular, the collection quantity of duodenal juice brarely exceeds 3.0 ml. Even when collection is continued over a longperiod of time, collection quantity exceeding 3.0 ml is not expected.Conversely, when excessive collection of duodenal juice b is continued,the mucosa of the duodenum 14 peels or becomes damaged. As a result,bleeding occurs particularly when a catheter generating negativepressure is used. Usefulness as a specimen is lost. Therefore, thecollection quantity of duodenal juice b is 3.0 ml or less, preferably1.0 ml or less, and more preferably 0.5 ml or less. The collectionquantity can be restricted by the shape or function of the samplingequipment.

In patients in which the pancreatic duct 11 or the bile duct isconstricted by the formation of a tumor, very little duodenal juice bmay be secreted. In this instance as well, if the collection quantity ofthe duodenal juice b is 0.001 ml, the duodenal juice b can be examinedusing an essentially immunological method.

As a result of the collection quantity of duodenal juice b beingrestricted in this way, composition of the collected duodenal juice bcan be kept constant. Detection accuracy can be maintained.

The collection quantity of duodenal juice b can be visibly confirmed.For example, when the catheter is used, the collection quantity of thecollected duodenal juice b is confirmed using calibrations on thesyringe (or the collection bottle connected to a vacuum). A calibrationindicating only the requisite quantity (such as 0.5 ml) may be displayedin advance on the collection container for duodenal juice b. In somecases, duodenal juice b could not be completely aspirated into thecontainer. The duodenal juice b may remain in the catheter duringcollection. Therefore, an accurate collection quantity is preferablyconfirmed by occasionally raising the tip section of the catheter fromthe fluid surface, and pulling the fluid within the catheter and airinto the container during collection.

(6) Collection of the duodenal juice b is continued until the collectionquantity of duodenal juice meets the condition of the requisite quantity(such as 0.5 ml or more) set to 0.001 ml or more and 3.0 ml or less.However, if the collection time for duodenal juice b reaches fiveminutes after the start of collection, collection of the duodenal juiceb is stopped even when the collection quantity does not meet thecondition (Step 6).

Here, a practitioner or a caretaker can monitor the collection time forduodenal juice by measuring time from the start of collection ofduodenal juice b using a clock, a timer, or the like.

As a result of the collection time being restricted in advance in thisway, the duodenal juice b can be efficiently collected in a short amountof time.

In addition, for example, when ordinary routine upper gastrointestinalendoscopy and collection of duodenal juice b are combined, anesthesiamay wear off during observation of the gastrointestinal tract unless thecollection time for duodenal juice b is restricted. The quantity ofanesthetic to be administered becomes difficult to determine. As aresult of the collection time being restricted in advance in this way,collection of the duodenal juice b and observation of thegastrointestinal tract can be simultaneously performed during thelimited amount of time the patient is in the anesthetized state.

(7) The interior of the stomach is observed using the endoscope insertedinto the stomach (Step 7).

Here, ordinary observation can be performed after the duodenal juice bis collected.

As described above, in the method of collecting a specimen according tothe present embodiment, duodenal juice b of a quality and a quantityenabling examination for upper digestive system disease can be collectedin a short amount of time. Furthermore, duodenal juice b allowing highdetection accuracy for upper digestive system disease can be obtained.

In the procedures of the method of collecting a specimen according tothe present embodiment, at Step 5, it is judged whether or not thecollection quantity of duodenal juice b meets the condition of therequisite quantity set to 0.001 ml or more and 3.0 ml or less. However,this condition is not required. A procedure is possible in which only acondition for collection time is prescribed, without setting thecollection quantity of duodenal juice b. The condition is thatcollection of the duodenal juice b is stopped within five minutes fromthe start of collection. More preferably, collection of the duodenaljuice b may be stopped within two minutes. When collection is performedfor two minutes, about 0.5 ml can be collected in most patients. Peelingof mucosa, bleeding, and the like which occur when collection isperformed for five minutes or more can be prevented. All that isrequired is that the requisite quantity for analysis of the specimen iscollected.

[Collection System for Specimen]

An example of a collection system for a specimen will hereinafter bedescribed. The method of collecting a specimen according to the presentembodiment is implemented in the collection system.

As shown in FIG. 4, a collection system 17 for a specimen includessampling equipment 18, a sensor 19, and a timer 20. The samplingequipment 18 collects the duodenal juice b and stores the collectedduodenal juice b. The sensor 19 detects the collection quantity of theduodenal juice b. The timer 20 detects the collection time for theduodenal juice b. The collection system 17 also includes a controller 21and an observation device (not shown). The controller 21 controls thestart and end of collection of the duodenal juice b by the samplingequipment 18 based on signals from the sensor 19 and the timer 20.

The sampling equipment 18 includes a catheter 22 for collection, acollection bottle 23, and a vacuum pump 24. The collection bottle 23 isattached to the base side of the catheter 22. The protease inhibitor isadded into the collection bottle 23 in advance.

For example, the ERCP cannula PR-130Q (manufactured by Olympus MedicalSystems Corporation) shown in FIG. 1 is used as the catheter 22 forcollection. The ERCP cannula PR-130Q is provided with two holes 25 inthe radial direction on the tip side. The sensor 19 is a fluid leveldetection sensor that detects the level of the duodenal juice b led intothe collection bottle 23. The sensor 19 detects whether or not thecollection quantity of the duodenal juice b is within a range of 0.001ml or more and 3.0 ml or less. The sensor 19 then sends a signal to thecontroller 21.

The timer 20 counts five minutes from the start of collection of theduodenal juice b and sends a signal to the controller 21.

The observation device (not shown) includes an endoscope and an imagemonitor.

In the collection system 17 for a specimen, first, the endoscope isadvanced to the duodenum of the subject. Confirmation is obtainedvisually (via the camera connected to the endoscope) using the monitor.The catheter 22 for aspiration is then inserted into the forcepsinsertion opening of the endoscope and projected outward from the tipsection of the endoscope. Next, the controller 21 sends signalsrespectively to the vacuum pump 24, the sensor 19, and the timer 20.

When the controller 21 sends a signal to the vacuum pump 24, collectionof the duodenal juice b is started as a result of startup of the vacuumpump 24. The duodenal juice b is led into the collection bottle 23. Whenthe controller 21 sends a signal to the sensor 19, the sensor 19 detectsthe fluid level. When the collection quantity of the duodenal juice bmeets the condition, the sensor 19 sends a signal to the controller 21.When the controller 21 sends a signal to the timer 20, the timer 20counts five minutes from the start of collection of the duodenal juice band sends a signal to the controller 21. When the sensor 19 or the timer20 sends a signal to the controller 21, the controller 21 sends a stopsignal to the vacuum pump 24 and stops the collection of the duodenaljuice b.

In this way, the duodenal juice b is collected using a collection systemfor a specimen in which a part of the method of collecting a specimenaccording to the present embodiment is automated. As a result,examination can be simplified. Moreover, the burden placed on theoperator can be reduced.

[Method of Diagnosing Subject]

Next, a method of diagnosing a subject to detect upper digestive systemdisease according to the present embodiment will be described.

Here, measurement of protein concentration in the duodenal juice b andcytological diagnosis are performed using the duodenal juice obtained bythe above-described method of collection of a specimen shown in FIG. 2.Diagnosis of a subject is thereby performed.

(Measurement of Protein Concentration)

Any type of protein is applicable as long as the concentration thereofcan be measured in the duodenal juice b and the concentration thereofchanges in relation to the upper digestive system disease. Specifically,the protein may be, for example, carcinoembryonic antigen (CEA)(carbohydrate chain binding form giant tumor marker protein), S100P(calcium binding protein), or IL-8 (cytokine). When pure pancreaticjuice c directly collected from the pancreatic duct is examined, theseproteins are known to be overexpressed in pancreatic cancer patients.The CEA, S100P, and IL-8 are proteins having different sizes, andstructural and functional characteristics. However, it has become clearin the present study that these proteins are all detected in theduodenal juice b containing pancreatic juice c. In addition, correlationbetween the protein concentration detected from the naturally secretedduodenal juice b containing pancreatic juice c, and pancreatic cancerand IPMN has also been confirmed. From these results, it is inferredthat the proteins reportedly detected in the pure pancreatic juice c canbe measured in the duodenal juice b collected using the method ofcollection of a specimen according to the present embodiment.

Furthermore, examination may be conducted using at least one proteinselected from the CEA, S100P, and IL-8. Alternatively, examination maybe conducted using a combination of two or more proteins. Detectionaccuracy can be increased by conducting examination using a combinationof a plurality of proteins.

The method of measuring the concentration of protein in the duodenaljuice b is merely required to be a common measuring method usingantigen-antibody reaction. For example, the ELISA method, achemiluminescence immunoassay method, a latex-coagulation method, or aimmunochromatographic method may be used. In addition, as a measuringmethod that does not use antigen-antibody reaction, measurement can betaken by comparison of peaks and spots through time-of-flight massspectrometry (TOF-MS) or two-dimensional electrophoresis.

In particular, the immunochromatographic method (theimmunochromatography device), among the above-described methods ofmeasuring protein, is preferably used. As a result, examination can beconducted easily and quickly in the endoscopy unit using a smallquantity of duodenal juice b. In addition, the patient can be promptlyinformed of the result of the examination. The subsequent treatment plancan be decided on the spot.

(Immunochromatography)

The immunochromatography (the immunochromatography device) willhereinafter be described with reference to FIG. 5A and FIG. 5B.

As shown in FIG. 5A, an immunochromatography device 26 is basicallycomposed of a detection unit 27 and a plastic case 28. The plastic case28 covers the detection unit 27.

As shown in FIG. 5B, the detection unit 27 has a carrier 29 as a base.The subject fluid is spread over the carrier 29. A sample pad 30 isdisposed on one end of the carrier 29. An absorbent pad 31 is disposedon the other end of the carrier 29. A linear first binding area 29 aholding a first antibody is provided in the carrier 29. A linear secondbinding area 29 b in which a second antibody is solid-phased is providedin a position at a distance from the first binding area 29 a. As shownin FIG. 5A, a sight dome 32 is opened in a position corresponding to thesample pad 30 in the case 28. An observation window 33 is opened in aposition corresponding to the second binding area 29 b provided in thecarrier 29.

The carrier 29 is merely required to be that on which the subject fluidcan be spread. For example, a nitrocellulose membrane filter issuitable.

The first antibody of the first binding area 29 a and the secondantibody of the second binding area 29 b are antibodies that recognizemutually differing epitopes of the protein to be detected. The firstantibody is labeled by a substance having a predetermined color. Thesubstance is, for example, a metal colloid, such as gold colloid orplatinum colloid, or a latex colloid (labeled substance) such assynthetic latex or natural rubber latex. The synthetic latex is, forexample, colored polystyrene latex. The labeled substance labeling thefirst antibody may be an enzyme or a radioactive material.

The sample pad 30 is composed of a sheet or film made of a materialhaving excellent absorption. The material is, for example, as a poroussynthetic resin, such as porous polyethylene or porous polypropylene.Alternatively, the sample pad 30 is composed of, for example, cellulosepaper, such as filter paper or cotton cloth, woven fabric, or nonwovenfabric.

The absorbent pad 31 is composed of a material capable of quicklyabsorbing and holding liquid. The material is, for example, a cottoncloth, a filter paper, or a porous plastic nonwoven fabric such as thatmade of polyethylene or polypropylene.

The immunochromatography device 26, configured as described above, isused as follows. The operator first drops the subject fluid from thesight dome 32 onto the sample pad 30 (or may soak the sample pad 30 inthe subject fluid). The dropped subject fluid spreads by moving over thecarrier 29, from the sample pad 30 towards the absorbent pad 31. Duringspreading, the target protein in the subject fluid binds with the firstantibody in the binding area 29 a and forms a complex. Then, the complexbinds with the second antibody in the second binding area 29 b and isabsorbed in the second binding area 29 b. As a result of accumulation ofthe complex in the second binding area 29 b, the second binding area 29b is colored a predetermined color. Therefore, the operator can measurethe presence or the concentration of the target protein by whether ornot a band of the predetermined color appears in the observation window33, or the darkness of the color of the band.

Here, when the collected duodenal juice b is added from the sight window32 of the immunochromatography device 26, the result can be obtained inthe observation window 33 in about 15 minutes. As a result, a specialexamination room or an examination device is not required. The result ofthe examination can be quickly obtained.

(Cytological Diagnosis)

To perform cytological diagnosis, the above-described protease inhibitoris preferably mixed with the collected duodenal juice in advance. As aresult of the protease inhibitor being added, pancreatic enzymeactivation is inhibited. Dissolution of cells in the pancreatic duct andthe bile duct can be suppressed, and cell shape can be preserved.Cytological diagnosis of the duodenal juice b is performed using amethod similar to that of ordinary cytological diagnosis of pancreaticjuice c and bile d. Specifically, the duodenal juice b is centrifugallyseparated. The precipitate cells are then placed on a slide glass andfixed with alcohol. Next, the cells are Papanicolaou-stained, and thecell shapes are observed under a microscope.

Determination in cytological diagnosis is performed in five stages:Class I (no atypical cells found); Class II (atypical cells found but nosuspicion of malignancy); Class III (atypical cells found but malignancyinconclusive); Class IV (atypical cells strongly suspected of malignancyfound); and Class V (atypical cells concluded to be malignant).

In pancreatic cancer, bile duct cancer, and gallbladder cancer, evidenceof adenocarcinoma cells are possibly seen (Class IV to V). Thecytological diagnosis can be used for definite diagnosis and screeningfor pancreatic cancer, bile duct cancer, and gallbladder cancer.

Example 1 to Example 6 will hereinafter be described.

Example 1

An upper gastrointestinal endoscope was inserted into 38 upper digestivesystem disease patients (32 pancreatic cancer patients and 6 controlgroup patients [2 IPMN patients, 1 pancreatic endocrine tumor (NET)patient, 1 MCN patient, 1 SCN patient, and 1 retention cyst patient]:diagnostic results based on postoperative pathological diagnosticresults) after fasting overnight. Gastric juice c was aspirated fromeach patient. When the endoscope reached the duodenum 14, a catheter 2for aspiration (PR-130Q manufactured by Olympus Medial SystemsCorporation) was inserted into the forceps insertion opening of theendoscope and projected outward from the tip section of the endoscope.The duodenal juice b was present in a plurality of sites in the secondposition 14X and the third position 14Y of the duodenum 14. The duodenaljuice b was collected from the tip side of the catheter 2. At this time,a 20 ml syringe 3 was connected to the base side of the catheter 2.

When collecting the duodenal juice b, the collection start time wasclocked using a timer. The duodenal juice b was collected for 2.5minutes. Subsequently, the syringe was exchanged with a new syringe.

The collected duodenal juice was immediately frozen for preservation at−80° C.

Next, the 38 patients were administered secretin. Collection of duodenaljuice b was started immediately after administration of secretin. Thesyringe was exchanged every 2.5 minutes. The collection quantity ofduodenal juice b was checked. Subsequently, the collected duodenal juiceb was immediately frozen for preservation at −80° C.

Furthermore, after the duodenal juice b had been frozen and thawed, CEAconcentration in the duodenal juice b was measured using an ELISA kit(manufactured by IBL Co., Ltd.).

As shown in FIG. 6, the average collection quantity of the duodenaljuice b is about 2.1 ml. The duodenal juice b is collected within 2.5minutes after administration of secretin from the 38 upper digestivesystem disease patients. On the other hand, the average collectionquantity of the naturally secreted duodenal juice b (zero minutes)collected in 2.5 minutes is about 1.3 ml. The average collectionquantity of naturally secreted duodenal juice b is slightly lesscompared to that of the control group that has been administeredsecretin. However, the quantity is sufficient for a specimen. As aresult, it has been found that about 1.0 ml to 2.0 ml of naturallysecreted duodenal juice b can be collected by collection performed forabout 2 to 3 minutes.

Next, CEA concentrations accompanying the elapse of time were analyzedfor each case of the patients. The results are shown in FIG. 7 as abox-plot. Malignant indicates pancreatic cancer. Benign indicates thecontrol group.

As shown in FIG. 7, the CEA concentration in the naturally secretedduodenal juice b (zero minutes) is higher than the CEA concentration inthe duodenal juice b after administration of secretin. It has been foundthat detection can be performed with the highest accuracy.

Furthermore, detection sensitivity (sensitivity of CEA) for pancreaticcancer was calculated from a cut-off value calculated from the controlgroup. In the calculation method, the highest CEA concentration amongthe CEA concentrations of the duodenal juices b of benign diseasesserves as a reference value (cut-off value). The result is determined tobe positive when the reference value is exceeded. The percentage of thenumber of patients to be positive in relation to the number ofpancreatic cancer subjects is determined. The sensitivity (%) of CEA topancreatic cancer is calculated. The sensitivity of CEA to pancreaticcancer per collection fraction is shown in Table 1.

TABLE 1 Elapsed Time From The Start Of Collection Cut-Off Of DuodenalJuice Value Sensitivity Specificity (minutes) (ng/ml) (%) (n) (%) (n) 0126 70 21/30 83 5/6 2.5~5.0 49 84 26/31 80 4/5  7.5~10.0 21 88 28/32 835/6 10.0~15.0 40 58 15/26 80 4/5

As shown in Table 1, the sensitivity of the naturally secreted duodenaljuice b (zero minutes) has been found to be about the same as that ofthe duodenal juice b after secretin stimulation. In addition, even whenthe duodenal juice b is continuously collected at 2.5-minute intervals,changes in the CEA concentration pattern and the sensitivity of eachpatient per fraction could not be seen. Therefore, it has been foundthat detection accuracy can be achieved by the naturally secretedduodenal juice b (zero minutes), and that the detection accuracy isabout the same as the sensitivity to pancreatic cancer of the duodenaljuice b obtained by being collected continuously for 10 minutes.

In measurement of the CEA concentration, no difference could be seendepending on whether or not a protease inhibitor had been added.

Example 2

In the present example, among the patients with upper digestive systemdisease described in Example 1, 31 cases of pancreatic disease patients(27 cases of pancreatic cancer and remaining cases IPMN and NET), and 1case each of MCN, SCN, and retention cyst were subjected to examination.The effects of addition of a protease inhibitor were studied.

Here, the base side of the catheter was bifurcated. The catheter wasconnected to two syringes (20 ml). One syringe was added with a proteaseinhibitor (Complete manufactured by F. Hoffmann-La Roche Ltd.) inadvance. The other syringe was not added.

The duodenal juice b was collected by aspiration for 2.5 minutes each,before and after administration of secretin.

In the syringe in which the protease inhibitor had been added, when thecollected duodenal juice b was led into the syringe, the proteaseinhibitor is degraded and mixed with the duodenal juice b.

The S100P concentration in the collected duodenal juice b wassubsequently measured using the ELISA method (manufactured by Cylex Co.,Ltd.). The results are shown in FIG. 8A and FIG. 8B as a box-plot. Thesensitivity (%) of S100P to pancreatic cancer and IPMN is shown in Table2. In FIG. 8A and FIG. 8B, malignant indicates pancreatic cancer. Benignindicates the control group.

TABLE 2 Elapsed Time From The Start of Collection Of Duodenal Cut-OffJuice Value Sensitivity Specificity (minutes) Inhibitor (pg/ml) (%) (n)(%) (n) 0 (−) 1518 53 16/30 100 6/6 2.5~5.0 0 37 11/30 80 4/5  7.5~10.00 42 13/31 100 6/6 0 (+) 3264 92 24/26 80 4/5 2.5~5.0 1500 85 23/27 804/5  7.5~10.0 7384 50 13/26 80 4/5

As shown in FIG. 8A and FIG. 8B, it has been found that the S100Pconcentration in the duodenal juice b is detected to be higher when theprotease inhibitor is added, compared to when the protease inhibitor isnot added. In addition, as shown in Table 2, the sensitivity of thenaturally secreted duodenal juice (zero minutes) is slightly lower whenthe protease inhibitor is added, compared to when the protease inhibitoris not added. However, in the subsequent 2.5-minute to 5.0 minutecollection fraction, the sensitivity increases by almost two times whenthe protease inhibitor is added, compared to when the protease inhibitoris not added. As a result, it has been found that, when the S100Pprotein concentration in the duodenal juice b is measured, detectionaccuracy increases as a result of the duodenal juice b being collectedwith the syringe serving as the collection container having a proteaseinhibitor added in advance.

Example 3

In the present example, the IL-8 concentration in the duodenal juice bwas measured using an ELISA kit (manufactured by R&D Systems Inc.).Here, the duodenal juice b was collected using the same method as thatdescribed in Example 2. The collected duodenal juice b was used as thespecimen.

The results are shown in FIG. 9A and FIG. 9B as a box-plot. Thesensitivity (%) of IL-8 to pancreatic cancer is shown in Table 3.Malignant indicates pancreatic cancer. Benign indicates the controlgroup. The cut-off value was calculated from the control group.

TABLE 3 Elapsed Time From The Start of Collection Of Duodenal Cut-OffJuice Value Sensitivity Specificity (minutes) Inhibitor (pg/ml) (%) (n)(%) (n) 0 (−) 1 47 14/30 83 5/6 2.5~5.0 1.5 37 11/30 80 4/5  7.5~10.0 228  9/32 83 5/6 0 (+) 4 68 17/25 80 4/5 2.5~5.0 2 59 16/27 100 5/5 7.5~10.0 1 62 16/26 67 4/6

As shown in FIG. 9A and FIG. 9B, it is has been found that the IL-8concentration in the duodenal juice b significantly increases in thenaturally secreted duodenal juice b and the duodenal juice b afteradministration of secretin when the protease inhibitor is added,compared to when the protease inhibitor is not added. In addition, asshown in Table 3, when the protease inhibitor is not added, thesensitivity of the IL-8 in the naturally secreted duodenal juice b topancreatic cancer is zero (%). It is thought that, in the instance ofthe naturally secreted duodenal juice b, the IL-8 is broken down andcannot be measured unless the protease inhibitor is added.

As a result, it has been found that, when the IL-8 concentration ismeasured as well, detection accuracy increases as a result of theduodenal juice b being collected with the syringe being added with aprotease inhibitor in advance.

Example 4

In the present example, the CEA concentration in the duodenal juice bwas checked using the immunochromatography method.

Here, as an immunochromatography device, a commercially available CEAimmunochromatographic examination equipment (Lana Mammo Card CEAmanufactured by Nippon Kayaku Co., Ltd.) was used. As the specimen,naturally secreted duodenal juice b (zero minutes) collected using thesame method as that in Example 1 was used.

Three microliters of the naturally secreted duodenal juice b (zerominutes) was dropped onto the CEA immunochromatographic examinationequipment. After the elapse of about 15 minutes, the CEA concentrationwas measured. The CEA concentration can be measured through comparisonwith an attached color chart.

As a result, the CEA concentration (about 100 ng/ml, and about 400ng/ml) inferred from the color chart was correlated with the CEAconcentration measured using the ELISA method. Therefore, it has beenfound that the CEA concentration in the duodenal juice b can bedetermined by the immunochromatography method.

Example 5

In the present example, cytological diagnosis of the duodenal juice bwas performed.

Here, as the specimen, naturally secreted duodenal juice b (zerominutes) collected using the same method as that in Example 2 was used.Cytological diagnosis was performed for both when the protease inhibitoris added and when the protease inhibitor is not added.

First, the collected duodenal juice b was centrifugally separated at3000 rpm for 10 minutes using a centrifuge (manufactured by Tomy SeikoCo., Ltd.). The precipitate cells were then applied onto a slide glassand fixed with alcohol. Subsequently, the cells werePapanicolaou-stained, and the cell shapes were observed under amicroscope.

As a result, the cell shapes were deformed in the fraction in which theprotease inhibitor was not added. Adenocarcinoma cells could not beidentified. Conversely, adenocarcinoma cells retaining cell shape couldbe identified in the fraction in which the protease inhibitor was added.In the pancreatic cancer patients, cells ranging from Class IV to ClassV were detected.

Example 6

Naturally secreted duodenal juice was collected from one bile ductcancer patient and one liver cancer patient. CEA concentrations of thesamples were measured using the same method as that in Example 1 wasused.

Duodenal juice CEA concentration for bile duct cancer patient was 190ng/ml and that for liver cancer patient was 241 ng/ml. Those CEAconcentrations were higher than a cut-off value shown in Example 1. As aresult, these tests for bile duct cancer and liver cancer weredetermined to be positive.

As described above, pancreatic diseases can be detected even whennaturally secreted duodenal juice b collected over a short period oftime is used, without administering a stimulant of pancreatic juicesecretion (such as secretin) and collecting duodenal juice b over a longperiod of time as in the conventional methods. The collected duodenaljuice b has been found to sufficiently serve as a specimen forpancreatic cancer detection.

While there has been described what is at present considered to be theseembodiments of the invention, it will be understood that variousembodiments which are not described yet may be made therein, and it isintended to cover all claims within the true spirit and scope of theinvention.

What is claimed is:
 1. A method of collecting a specimen to detect upperdigestive system disease, wherein the method includes: a step ofpositioning sampling equipment in the duodenum of the subject into whichduodenal juice is secreted, the sampling equipment being used to collectand store the duodenal juice; a step of collecting duodenal juicesecreted in the duodenum using the sampling equipment withoutadministering a stimulant of pancreatic juice secretion or a stimulantof bile secretion to the subject; and a step of stopping collection ofthe duodenal juice when collection quantity of the duodenal juicereaches a predetermined quantity which is 3.0 ml or less.
 2. The methodof collecting a specimen to detect upper digestive system diseaseaccording to claim 1, wherein the collecting of the duodenal juice isstopped within five minutes from the start of collection.
 3. The methodof collecting a specimen to detect upper digestive system diseaseaccording to claim 1, wherein the upper digestive system disease ispancreaticobiliary disease.
 4. The method of collecting a specimen todetect upper digestive system disease according to claim 1, wherein themethod including a step of adding a stabilizer for duodenal juice inadvance into a collection container serving as the sampling equipment,and the collected duodenal juice secreted in the duodenum using thesampling equipment is stored within the collection container.
 5. Themethod of collecting a specimen to detect upper digestive system diseaseaccording to claim 1, wherein the method includes a step of aspiratingresidual liquid present within the stomach of the subject before thestep of positioning the sampling equipment in the duodenum of thesubject.
 6. The method of collecting a specimen to detect upperdigestive system disease according to claim 1, wherein the methodincludes a step of observing the interior of the stomach by anobservation device inserted into the stomach after the duodenal juice iscollected by the sampling equipment.
 7. A method of diagnosing a subjectto detect upper digestive system disease, wherein the method includes: astep of positioning sampling equipment in the duodenum of the subjectinto which duodenal juice is secreted, the sampling equipment being usedto collect and store the duodenal juice; a step of collecting duodenaljuice secreted in the duodenum using the sampling equipment withoutadministering a stimulant of pancreatic juice secretion or a stimulantof bile secretion to the subject; a step of stopping collection of theduodenal juice when collection quantity of the duodenal juice reaches apredetermined quantity which is 3.0 ml or less; and a step of detectingconcentration of a protein included in the collected duodenal juiceusing an immunochromatography method.
 8. The method of diagnosing asubject to detect upper digestive system disease according to claim 7,wherein the protein is at least one or a combination of a plurality ofproteins selected from CEA, S100P, and IL-8.
 9. The method of diagnosinga subject to detect upper digestive system disease according to claim 7,wherein the upper digestive system disease is pancreaticobiliarydisease.
 10. A method of diagnosing a subject to detect upper digestivesystem disease, wherein the method includes: a step of positioningsampling equipment in the duodenum of the subject into which duodenaljuice is secreted, the sampling equipment being used to collect andstore the duodenal juice; a step of collecting duodenal juice secretedin the duodenum using the sampling equipment without administering astimulant of pancreatic juice secretion or a stimulant of bile secretionto the subject; a step of stopping collection of the duodenal juice whencollection quantity of the duodenal juice reaches a predeterminedquantity which is 3.0 ml or less; and a step of conducting examinationfor cytological diagnosis of the collected duodenal juice.
 11. A methodof collecting a specimen to detect upper digestive system disease,wherein the method includes: a step of positioning sampling equipment inthe duodenum of the subject into which duodenal juice is secreted, thesampling equipment being used to collect and store the duodenal juice; astep of collecting duodenal juice secreted in the duodenum using thesampling equipment without administering a stimulant of pancreatic juicesecretion or a stimulant of bile secretion to the subject; and a step ofstopping collection of the duodenal juice within five minutes from thestart of collection.
 12. The method of collecting a specimen to detectupper digestive system disease according to claim 11, wherein the upperdigestive system disease is pancreaticobiliary disease.
 13. The methodof collecting a specimen to detect upper digestive system diseaseaccording to claim 11, wherein the method includes a step of adding astabilizer for duodenal juice in advance into a collection containerserving as the sampling equipment, and the collected duodenal juicesecreted in the duodenum using the sampling equipment is stored withinthe collection container.
 14. The method of collecting a specimen todetect upper digestive system disease according to claim 11, wherein themethod includes a step of aspirating residual liquid present within thestomach of the subject before the step of positioning the samplingequipment in the duodenum of the subject.
 15. The method of collecting aspecimen to detect upper digestive system disease according to claim 11,wherein the method includes a step of observing the interior of thestomach by an observation device inserted into the stomach after theduodenal juice is collected by the sampling equipment.
 16. A method ofdiagnosing a subject to detect upper digestive system disease, whereinthe method includes: a step of positioning sampling equipment in theduodenum of the subject into which duodenal juice is secreted, thesampling equipment being used to collect and store the duodenal juice; astep of collecting duodenal juice secreted in the duodenum using thesampling equipment without administering a stimulant of pancreatic juicesecretion or a stimulant of bile secretion to the subject; a step ofstopping collection of the duodenal juice within five minutes from thestart of collection; and a step of detecting concentration of a proteinincluded in the collected duodenal juice using an immunochromatographymethod.
 17. The method of diagnosing a subject to detect upper digestivesystem disease according to claim 16, wherein the protein is at leastone or a combination of a plurality of proteins selected from CEA,S100P, and IL-8.
 18. A method of diagnosing a subject to detect upperdigestive system disease, wherein the method includes: a step ofpositioning sampling equipment in the duodenum of the subject into whichduodenal juice is secreted, the sampling equipment being used to collectand store the duodenal juice; a step of collecting duodenal juicesecreted in the duodenum using the sampling equipment withoutadministering a stimulant of pancreatic juice secretion or a stimulantof bile secretion to the subject; a step of stopping collection of theduodenal juice within five minutes from the start of collection; and astep of conducting examination for cytological diagnosis of thecollected duodenal juice.
 19. The method of collecting a specimen todetect upper digestive system disease according to claim 1, wherein thesampling equipment is positioned in the duodenum in a position furtherdistal from a stomach than a papilla of Vater.
 20. The method ofdiagnosing a subject to detect upper digestive system disease accordingto claim 7, wherein the sampling equipment is positioned in the duodenumin a position further distal from a stomach than a papilla of Vater. 21.The method of diagnosing a subject to detect upper digestive systemdisease according to claim 10, wherein the sampling equipment ispositioned in the duodenum in a position further distal from a stomachthan a papilla of Vater.
 22. The method of collecting a specimen todetect upper digestive system disease according to claim 11, wherein thesampling equipment is positioned in the duodenum in a position furtherdistal from a stomach than a papilla of Vater.
 23. The method ofdiagnosing a subject to detect upper digestive system disease accordingto claim 16, wherein the sampling equipment is positioned in theduodenum in a position further distal from a stomach than a papilla ofVater.
 24. The method of diagnosing a subject to detect upper digestivesystem disease according to claim 18, wherein the sampling equipment ispositioned in the duodenum in a position further distal from a stomachthan a papilla of Vater.
 25. The method of collecting a specimen todetect upper digestive system disease according to claim 1, wherein thesampling equipment is positioned in the duodenum in a position between astomach and a papilla of Vater.
 26. The method of diagnosing a subjectto detect upper digestive system disease according to claim 7, whereinthe sampling equipment is positioned in the duodenum in a positionbetween a stomach and a papilla of Vater.
 27. The method of diagnosing asubject to detect upper digestive system disease according to claim 10,wherein the sampling equipment is positioned in the duodenum in aposition between a stomach and a papilla of Vater.
 28. The method ofcollecting a specimen to detect upper digestive system disease accordingto claim 11, wherein the sampling equipment is positioned in theduodenum in a position between a stomach and a papilla of Vater.
 29. Themethod of diagnosing a subject to detect upper digestive system diseaseaccording to claim 16, wherein the sampling equipment is positioned inthe duodenum in a position between a stomach and a papilla of Vater. 30.The method of diagnosing a subject to detect upper digestive systemdisease according to claim 18, wherein the sampling equipment ispositioned in the duodenum in a position between a stomach and a papillaof Vater.